DNA Repair, Genome Stability and Aging

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Cell, Vol. 120, 497512, February 25, 2005, Copyright 2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.01.028

ReviewDNA Repair, GenomeStability, and Aging

David B. Lombard,1,2 Katrin F. Chua,1 ies per cell. Many mutants with phenotypes that resem-

ble premature aging possess defects in nuclear DNARaul Mostoslavsky,

1

Sonia Franco,

1

Monica Gostissa,1 and Frederick W. Alt1,* repair. Therefore, we focus on the role of nuclear DNA

damage in mammalian aging, emphasizing recent work1Howard Hughes Medical Institute

The Childrens Hospital in mouse models.

Department of Genetics

Harvard Medical School and DNA Damage, Reactive Oxygen Species, and AgingThe CBR Institute for Biomedical Research A large body of evidence argues that DNA damage andBoston, Massachusetts 02115 mutations accumulate with age in mammals (Vijg,2Department of Pathology 2000). Cells harboring mutations at defined loci haveBrigham and Womens Hospital been shown to increase with age in humans and mice.Boston, Massachusetts 02115 Cytogenetically visible lesions such as translocations,

insertions, dicentrics, and acentric fragments also ac-

cumulate in aging mammalian cells. Mice with inte-

grated reporter arrays have allowed estimates of theAging can be defined as progressive functional de-

age-related occurrence of both point mutations andcline and increasing mortality over time. Here, we re- larger genomic rearrangements. Such analyses haveview evidence linking aging to nuclear DNA lesions: revealed considerable variation in mutation spectra be-DNA damage accumulates with age, and DNA repair tween different tissues. These differences likely reflectdefects can cause phenotypes resembling premature functional characteristics of those tissues, such as mi-aging. We discuss how cellular DNA damage re- totic rate, transcriptional activity, metabolism, and thesponses may contribute to manifestations of aging. action of specific DNA repair systems.We review Sir2, a factor linking genomic stability, me- Reactive Oxygen Species: An Important Sourcetabolism, and aging. We conclude with a general dis- of Age-Related DNA Damagecussion of the role of mutant mice in aging research There are many sources of DNA damage. In addition toand avenues for future investigation. external sources, such as ionizing radiation and geno-

toxic drugs, there are also cell-intrinsic sources, suchIntroduction as replication errors, spontaneous chemical changes toAlthough aging is nearly universally conserved among the DNA, programmed double-strand breaks (DSBs) (ineukaryotic organisms, the molecular mechanisms un-

lymphocyte development), and DNA damaging agentsderlying aging are only beginning to be elucidated. A that are normally present in cells. The latter categoryuseful conceptual framework for considering the prob- includes reactive oxygen species (ROS), such as super-lem of aging is the Disposable Soma model (Kirkwood oxide anion, hydroxyl radical, hydrogen peroxide, nitricand Holliday, 1979). This model proposes that organ- oxide, and others. Major sources of cellular ROS pro-isms only invest enough energy into maintenance of the duction are the mitochondria, peroxisomes, cytochromesoma to survive long enough to reproduce. Aging oc- p450 enzymes, and the antimicrobial oxidative burst ofcurs at least in part as a consequence of this imperfect phagocytic cells. ROS can cause lipid peroxidation,maintenance, rather than as a genetically programmed protein damage, and several types of DNA lesions: sin-process. Although aging may involve damage to vari- gle- and double-strand breaks, adducts, and cross-ous cellular constituents, the imperfect maintenance of links. The situation in which ROS exceed cellular anti-nuclear DNA likely represents a critical contributor to oxidant defenses is termed oxidative stress. As normalaging. Unless precisely repaired, nuclear DNA damage byproducts of metabolism, ROS are a potential sourcecan lead to mutation and/or other deleterious cellular of chronic, persistent DNA damage in all cells and mayand organismal consequences. Damage to both nuclear

contribute to aging (Sohal and Weindruch, 1996). TheDNA, which encodes the vast majority of cellular RNA ROS theory of aging is discussed in depth in this issueand proteins, and mitochondrial DNA have been pro- of Cell by Balaban et al. (2005). In brief, longer-livedposed to contribute to aging (Karanjawala and Lieber, species generally show higher cellular oxidative stress2004). The reader is referred to the review by Balaban resistance and lower levels of mitochondrial ROS pro-et al. in this issue of Cell concerning the potential role duction than shorter-lived species. Caloric restriction,of mitochondrial DNA damage in aging (Balaban et al., an intervention that extends life span in many organ-2005). Nuclear DNA is an attractive target for aging- isms, likely decreases ROS production (Barja, 2004). Inrelated changes since it must last the lifetime of the model organisms, many mutations that promote lon-cell, unlike other cellular constituents, which can be re- gevity concomitantly increase oxidative stress resis-placed. In addition, the nuclear genome is present at tance (Finkel and Holbrook, 2000). In addition, levels ofonly two to four copies per cell, rendering it potentially 8-oxoguanine (oxo8dG), a major product of oxidativevery vulnerable to lesions; by contrast, the mito- damage to DNA, accumulate with age (Hamilton et al.,chondrial genome is present at several thousand cop- 2001).

The potential importance of oxidative damage toDNA in age-related dysfunction is highlighted by a re-*Correspondence: [email protected]


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Figure 1. Multiple Pathways to Senescence

These pathways are employed in a cell- and

organism-specific fashion. In MEFs, activa-

tion of p19ARF leads to stabilization of p53.

In certain human cell types (e.g., fibroblasts

and keratinocytes), telomere attrition can ac-

tivate p53 via the ATM and (potentially) ATRkinases. Senescence can also be triggered

in human cells via p16 expression (Itahana et

al., 2004).

Activated p53 induces senescence via a

complex gene expression program that in-

cludes induction of p21. The role of Rb in

senescence involves repression of E2F target

genes as well as alterations in chromatin

structure. Senescent cells may contribute to

aging via depletion of stem cell pools and/

or elaboration of factors that interfere with

tissue function. Factors elaborated by se-

nescent cells may also stimulate the growth

of epithelial tumors (Krtolica and Campisi,

2002).

cent study of postmortem human brain tissue (Lu et al., passage in culture, a process termed replicative senes-

cence. Replicative senescence has been employed as2004), which found that many nuclear genes involved

in critical neural functions show reduced expression af- a cellular model for aging; many mutations in DNA re-

pair genes that cause premature aging phenotypes alsoter age 40, concomitant with elevated levels of oxo8dG

and DNA damage in their promoters. However, the high confer premature replicative senescence (Table 1).

Replicative Senescence Differs between Humanlevels of oxidative DNA damage found by these investi-

gators is at odds with other much lower estimates of and Mouse Cells

In many primary human cell lines, replicative senes-the amount of oxidatively damaged DNA in cells (Hamil-

ton et al., 2001). This highlights the difficulty of accu- cence occurs secondary to attrition of the ends of the

chromosomes, the telomeres, in a process termed in-rately measuring ROS and oxidative damage experi-

mentally. Additional unresolved issues concerning the trinsic senescence (Itahana et al., 2004). In mammalian

cells, the ends of the chromosomes consist of a ter-ROS theory of aging exist that will require clarification.

If ROS are indeed an important source of aging-associ- minal 3#

single-stranded tail, the G strand overhang,which is buried into adjacent double-stranded repeti-ated damage, it is currently unclear whether nuclear or

mitochondrial DNA is the most relevant functional tive telomeric DNA, forming a protective t loop higher-

order structure (de Lange, 2002). This t loop is stabi-target (Barja and Herrero, 2000; Hamilton et al., 2001).

Among long-lived mutants, the correlation with oxida- lized by a D loop, or displacement loop: the region

formed between the invading end of the telomere intotive stress resistance is a frequent but not universal

one: in some mutants, longevity occurs despite un- adjacent double-stranded DNA. The G strand overhang

is the substrate for the enzyme telomerase, which em-changed resistance to ROS or other forms of stress.

Additionally, mice heterozygous for a mutation in the ploys an RNA template, Terc, to extend telomeres dur-

ing S phase, thereby countering the natural shorteningmitochondrial enzyme that processes superoxide, Sod2,

live out a normal lifespan, despite accumulating higher of telomeres that would otherwise occur with each cell

division. Telomerase access to its substrate is, in turn,levels of nuclear and mitochondrial 8oxodG with age

(Van Remmen et al., 2003). Thus, while several lines of regulated by telomeric proteins, which also modulate

the conformational changes required for telomere repli-evidence argue for important role for ROS in aging,

many questions remain regarding this hypothesis. In cation and subsequent reestablishment of a protectiveend structure. In many types of human cells, includingthis regard, it is possible that additional mutations may

be required to fully unveil potential effects of ROS fibroblasts, telomeres shorten with each successive

generation. Critically short telomeres trigger the onset(see below).

of senescence through a process that may involve loss

of the t loop structure and/or loss of protective proteinsCellular Senescence: A Link between Cellular(referred to as uncapping). Such uncapped telomeresDamage and Aging?are then recognized by the cell cycle checkpoint ma-ROS and many other DNA-damaging agents can causechinery as DNA damage, leading to cell cycle arrestcells to enter a state of irreversible cell cycle arrest ac-(Ben-Porath and Weinberg, 2004).companied by characteristic morphologic and func-

In contrast to primary human fibroblasts, which ex-tional alterations, termed senescence (Ben-Porath and

press very low levels of telomerase activity, MEFs de-Weinberg, 2004). The induction of senescence depends

rived from M. musculus possess long telomeres andon pathways involving the p53 and Rb proteins (Figure

easily detectable telomerase activity (Itahana et al.,1). Cellular senescence has been best characterized in

2004). Thus, MEFs ordinarily do not senesce due tocultures of human fibroblasts and mouse embryo fibro-blasts (MEFs), which cease expanding after repeated telomeric attrition. Instead, senescence of wild-type


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is altered by culture under different oxygen tensions,Table 2. Common Pathologic Features of Aging in Mice (after

and telomere shortening in these cells is itself ac- Bronson and Lipman [1991]; Cao et al. [2003a])celerated by elevated levels of oxidative stress. It is

Hyperplasia/Neoplasianow clear that senescence can be induced by a variety

Adrenal hyperplasiaof different types of cellular injury (Figure 1).AngiosarcomaCellular Senescence and AgingHarderian gland adenomaWhat is the relationship between cellular senescenceEndometrial hyperplasia

and aging? The evidence that cellular senescence ac- Lung adenomatually plays a role in aging is correlative: senescent cells Lymphoma

Mammary gland adenocarcinomaaccumulate in vivo in mammals with increasing age andMast cell tumorat sites of pathology (Itahana et al., 2004), and manyOvarian cystadenomamouse and human models of premature aging are ac-Paraovarian cyst

companied by premature cellular senescence in vitroPituitary adenoma

(Table 1). Of note, late-generation Terc-deficient mice Sarcomashow some signs of accelerated aging (Lee et al., 1998; Thyroid follicular cell hyperplasia

Uterine leiomyoma/leiomyosarcomaRudolph et al., 1999). Two general models have been

proposed to explain how cellular senescence may con- Leukocytic Infiltratestribute to aging (Krtolica and Campisi, 2002; Pelicci,

Kidney2004). First, senescence of progenitor or stem cells

Liverthemselves could impair tissue renewal. In this regard,

Lungthe Polycomb group repressor Bmi1 appears to control Mesentery/omentumPerineuriumlevels of hematopoietic stem cells via negatively regu-Salivary glandlating the induction of senescence specifically in these

Genitourinary systemstem cells. Second, senescent cells secrete proteases

and other factors that may disrupt tissue function. In Hydronephrosisthis regard, senescence has a complex relationship Ovarian/testicular atrophy

Seminal vesicle dilationwith neoplasia. Senescence has been postulated to oc-Renal tubular dilationcur as a tumor suppressor mechanism, whereby cells

that have undergone a genotoxic insult and therefore Bonepossess the potential for neoplastic transformation en- Decreased cancellous boneter a state in which they are incapable of dividing (Krtol- Degenerative joint disease

Molar teeth periodontitisica and Campisi, 2002). However, senescent stromalProliferations in the head/spinecells can actually promote the growth of epithelial can-

cers, malignancies that occur with increased incidenceNeurologicalin the elderly. Senescence has been offered as an ex-Hydrocephalus

ample of antagonistic pleiotropy, a process that is Neuronal lipofuscinosisbeneficial in young organisms but deleterious later in Radiculopathy

White matter gliosislife: senescence suppresses cancer by preventing po-

tentially tumorigenic cells from dividing but may poten- Othertially contribute to organ dysfunction in the aged

Amyloidosisthrough a variety of mechanisms, perhaps even con- Fatty change of the livertributing to neoplasia in this setting (Krtolica and Cam- Focal myocardial degeneration

Hepatocyte polyploidizationpisi, 2002). However, a causal relationship between cel-Thymic involutionlular senescence and organismal aging has yet to be

proved.

DNA Repair and Agingphenotypes that, in some respects, caricature aging.

The accurate maintenance of nuclear DNA is critical to For reference, the features of aging as it occurs in wild-cellular and organismal function, and therefore, numer-

type mice are enumerated in Table 2, and the featuresous DNA repair systems have evolved. There is some

of mouse DNA repair/metabolism mutants showingevidence that the intrinsic fidelity and activity of such

some features of aging are listed in Table 1. We nowsystems in different species may influence the rate of

turn to discussing specific gene defects and their rele-age-associated functional decline (Hart and Setlow,

vance to aging.1974), although such studies need to be performed with

modern methodology. As outlined below, the efficiency

of cellular DNA repair machinery itself may decline with The ATM-p53 Axis: A Link between the DNA

Damage Response and Aging?age. Many different types of DNA lesions exist. DNA

DSBs are repaired via the nonhomologous end-joining Unrepaired or improperly repaired DSBs have serious

potential consequences for the cell: cell death, senes-(NHEJ) and homologous recombination (HR) pathways,

whereas lesions on a single strand of DNA are repaired cence, dysregulation of cellular functions, genomic in-

stability, and, in higher organisms, oncogenic trans-via base excision repair (BER) and nucleotide excision

repair (NER) (and its subpathways). In mice and hu- formation. The initial step in DSB repair is detection ofthe lesion, and this step involves the ataxia-telangiecta-mans, mutations in certain DNA repair genes lead to


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protein although several possible mechanisms are con-

ceivable (see below). In this context, the phenotype of

ATM-deficient cells may be relevant. In particular, such

cells manifest sensitivity to DSB-inducing agents and

have marked genomic instability; they also grow poorly

and senesce prematurely in culture (Barzilai et al.,

2002).

Genomic instability or DNA repair defects of ATM de-

ficiency could contribute to the aging-like features of

this disorder (see below). In this context, the premature

cellular senescence phenotype of ATM deficiency is

rescued by p53 deficiency (Xu et al., 1998), suggesting

a role for ATM-independent, p53-dependent check-

point responses. By extension, such responses also

might contribute to premature-aging phenotypes asso-

ciated with ATM deficiency. Another potential function

for ATM in suppressing progeroid phenotypes may be

related to reported ATM functions in regulating intracel-

lular ROS levels and sensitivity to these molecules (Bar-

zilai et al., 2002; Ito et al., 2004). Persistently elevated

ROS levels in AT cells might cause chronic damage to

DNA and other cellular macromolecules. However,

given the lack of an AT-like phenotype of the Sod2+/

mouse, progeroid features of ATM deficiency likely re-

flect more than increased ROS levels. One possibilityFigure 2. A Highly Simplified View of the DNA Damage Responsewould be elevated ROS levels in conjunction with loss

See text for details.of another ATM function, such as ability to respond to

ROS-induced DNA damage. Finally, ATM plays an im-

portant role in telomere maintenance; AT cells showsia mutated (ATM) kinase, other related phosphatidyl-

shortened telomeres and an increased incidence of te-inositol 3-kinase-like kinases (PIKKs), and other pro-

lomeric fusions (Pandita, 2002). Moreover, mice lackingteins (Figure 2; Bassing and Alt, 2004). Activated ATM

both ATM and Terc have higher levels of telomeric dys-phosphorylates numerous proteins involved in the G1/S,

function than generation-matched Terc-deficient mu-intra-S, and G2/M checkpoint responses and addition-

tants, as well as proliferative defects in multiple tissues,

ally phosphorylates factors involved in DNA repair decreased survival, and clinical evidence of premature(Bassing and Alt, 2004).aging (Wong et al., 2003). Thus, human AT patients may

An important target of ATM is the p53 protein, whichshow aging-like effects, at least in part, as a conse-

plays a role in the cellular response to numerous geno-quence of telomeric dysfunction, an effect that ordinar-

toxic insults including DSBs. Phosphorylation of p53 byily may be masked in the mouse due to the long telo-

ATM and kinases downstream of ATM is a major mech-meres of this organism.

anism leading to upregulation of p53 levels and activity,The phenotypes of ATM deficiency are influenced by

although p53 also can be activated via ATM-indepen-environmental conditions. In the ATM-deficient mouse,

dent mechanisms. Activated p53 stimulates or repressesthe onset of T cell lymphoma can be delayed by treat-

transcription of many target genes and coordinatesment with an antioxidant, suggesting that dietary factors

checkpoint, senescence, and apoptosis pathways in impact on the expression of this condition (Schubert etresponse to DSBs and other signals. In this regard, p53, al., 2004). Neurological dysfunction in this model canthrough modulation of various downstream targets, similarly be ameliorated via antioxidant treatment (Brownetriggers arrest/senescence or apoptosis (Meek, 2004). et al., 2004). Additionally, the frequency of thymic lym-

The exact factors that determine the differential out- phoma varies among different strains of ATM-deficientcomes of this complex program are not yet completely mice, a finding that might reflect different backgroundelucidated but vary with the cell type, as well as the mutations and/or housing conditions and the types ofkind, intensity, and duration of the damage. pathogens present (Petiniot et al., 2002). These obser-ATM and Aging vations raise the possibility that other aspects of thePerturbations in ATM function can lead to symptoms of ATM-deficient phenotype, including the prematureaccelerated aging. Patients with mutations in the ATM aging observed in human patients, may be influencedgene suffer from Ataxia-Telangiectasia (AT), a condition by environment. This is a theme that we will return tocharacterized by a prematurely aged (progeroid) ap- in our discussion of other premature aging models.pearance, immunodeficiency, cerebellar degeneration, p53 and Agingand cancer (Shiloh and Kastan, 2001). ATM deficiency Various lines of evidence suggest that p53 plays op-in mice recapitulates many of these phenotypes, al- posing roles in the aging process. While p53 sup-though the progeroid features of the mouse models are presses the onset of malignancy and thereby extendsless prominent than in the human disease. Given the life span, at the same time it promotes cellular senes-

many targets of ATM, it is difficult to trace the progeroid cence and apoptosis in response to DNA damage, po-tentially contributing to the clinical changes of aging.appearance of AT patients to a specific function of this


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Figure 3. NHEJ at General DSBs

Factors associated with progeroid mutant

phenotypes (Ku80 and DNA-PKcs) are

shown in red. See text for details.

Thus, p53 function may display antagonistic pleiotropy Ku70, Ligase IV, and XRCC4) are conserved from yeast

to mammals; they are indispensable for all NHEJ reac-(Campisi, 2002). The role of p53 in aging cannot be di-

tions. In contrast, the other two NHEJ factors, DNA-rectly tested using p53-deficient mice, as such animals

PKcs (DNA-dependent protein kinase catalytic subunit)invariably die of malignancy before age-related changes

and Artemis, have evolved more recently and arebecome manifest. However, two mouse strains that ex-thought to be required for joining the subset of DNApress C-terminal p53 fragments along with full-length p53ends that require processing prior to ligation. Ku70 andhave been reported to show accelerated aging pheno-

Ku80 bind as a heterodimer to DSBs, where they aretypes and a lower incidence of malignancy (Maier et al.,thought to serve a protective function and to enlist2004; Tyner et al., 2002). It has been proposed thatother factors. In this regard, Ku70 and Ku80 recruitthese truncated p53 proteins exert their effects byDNA-PKcs, and together the three proteins form themodifying the activity of endogenous wild-type p53DNA-PK holoenzyme. DNA-PK activates Artemis, whichprotein. Only nonphysiologic activation of p53 leads tofunctions as an endonuclease to process ends thatprogeria, as mice expressing extra copies of wild-typecannot be directly rejoined. Finally, XRCC4 and Ligasep53 under the control of its own promoter do not showIV, which are likely recruited by Ku, function together tosigns of premature aging (Garcia-Cao et al., 2002).catalyze end ligation itself. NHEJ often occurs concom-Further arguing for a role for p53 in promoting aging,itant with loss of a few nucleotides at the site of joining.a null mutation in a gene functioning downstream of

NHEJ plays a critical role in general DNA DSB repairp53 in the induction of apoptosis, p66Shc, confers oxi-and, correspondingly, in the maintenance of genomicdative stress resistance and extends mouse life spanstability. In addition, NHEJ plays a role in repairing ge-(Migliaccio et al., 1999). The p66Shc protein shortensnetically programmed DSBs in the context of antigenmurine life span via at least two mechanisms: it in-

receptor variable region gene assembly (V(D)J recombi-creases constitutive intracellular ROS levels, and it pro-nation) in developing lymphocytes. Mice with targetedmotes cell death in response to oxidative stress. It isinactivating mutations in NHEJ genes display pheno-unclear how p66shc evolved to play such a role in thetypes that reflect loss of these functions (Ferguson andcell, since the shorter isoforms of this protein, p52 andAlt, 2001). All NHEJ-deficient mice suffer from severe

p46, fulfill an entirely dissimilar cellular function, trans-combined immunodeficiency as a consequence of an

ducing signals from tyrosine kinases to ras.inability to productively rejoin broken V(D)J gene seg-

ments in developing B and T cells. NHEJ deficiency isA Putative Role for NHEJ in Suppressing Aging also associated with ionizing radiation-sensitivity andNHEJ in DSB Repair and Telomere Maintenance an elevated incidence of spontaneous genomic insta-We now turn from a discussion of factors upstream of bility. MEFs deficient for XRCC4, Ligase IV, Ku70, orDNA repair to a consideration of repair pathways them- Ku80 (but not DNA-PKcs or Artemis) senesce prema-selves and their involvement in aging. NHEJ is one of turely in culture, and mice deficient for these fourthe two major DSB repair pathways in mammalian cells factors are very small and show widespread neuronal

(Bassing and Alt, 2004). NHEJ is mediated by at least apoptosis during embryogenesis. Premature senes-cence and neuronal apoptosis, but not small size, aresix core factors (Figure 3). Four of these proteins (Ku80,


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relieved by p53 deficiency. Thus, the former pheno- could directly impair cellular function by promoting

DNA deletions. In this context, Ku80-deficient mice aretypes occur as a response to, rather than as a direct

consequence of, unrepaired DSBs (Ferguson and Alt, found to have a low rate of mutations at a marker locus

(Rockwood et al., 2003); such an assay might not de-2001). Mice deficient in NHEJ and p53 invariably suc-

cumb to pro-B cell lymphomas as a consequence of tect large deletions or rearrangements, however. Alter-

natively, unrepaired DSBs could trigger elevated levelsaberrant repair of V(D)J recombination-associated

DSBs in developing B cells, leading to oncogenic trans- of cellular senescence and apoptosis. In this regard,

p53-dependent responses are critical in mediating thelocations (Bassing and Alt, 2004).

In addition to their roles in DSB repair, at least three premature senescence and neuronal apoptosis pheno-

types of certain NHEJ mutants (Ferguson and Alt,core NHEJ factors, namely Ku70, Ku80, and DNA-PKcs,

localize to telomeres (d'Adda di Fagagna et al., 2001). 2001).

Despite the phenotypes of the Ku80- and DNA-PKcs-Deficiency for any of these, as well as for Artemis

(Rooney et al., 2003), is associated with an increased deficient mice, several observations suggest that any

potential roles for NHEJ in suppressing aging might befrequency of end-to-end chromosomal fusions in MEFs,

suggesting a role for these proteins in chromosomal end more complicated. Ku70- and Artemis-deficient mice

thus far have not been reported to show prematurecapping. There is conflicting data on whether NHEJ

factors protect against telomere shortening. NHEJ also aging phenotypes. If Ku70-deficient mice actually lack

an aging phenotype, the apparent Ku80-deficient pro-promotes telomeric fusions in some circumstances. In-

creased telomere end-to-end fusions are observed in the geroid phenotype must be rationalized in the context

of the finding that targeted ablation of Ku70 results insetting of the telomeric attrition associated with Terc defi-

ciency or inhibition of TRF2, a protein thought to function dramatically reduced Ku80 levels (Gu et al., 1997). In

addition, aging-related phenotypes thus far have notin end capping, and these end-to-end fusions are elimi-

nated in NHEJ-deficient backgrounds (Espejel et al., been reported in several other independently generated

DNA-PKcs mutant mouse strains. Ligase IV- and XRCC4-2002a, 2002b; Smogorzewska et al., 2002). Thus, by li-

gating uncapped telomeres, NHEJ may actually pro- deficient mice show embryonic lethality due to wide-

spread neuronal apoptosis, precluding aging analyses.mote genomic instability. Aside from roles in the NHEJ

reaction and in telomere maintenance, Ku70, Ku80, However, the lack of a consistent aging-like phenotype

in other lines of long-lived Ku-, DNA-PKcs-, or Artemis-DNA-PKcs, and Artemis have other known or sus-

pected cellular functions, including DSB or checkpoint deficient mice is difficult to explain if NHEJ plays a gen-

eral role in delaying manifestations of aging (Karanjawalasignaling, whereas XRCC4 and Ligase IV appear to

function only in end-ligation during NHEJ. In summary, and Lieber, 2004). In this regard, it is conceivable that

some aging-related phenotypes might have beenNHEJ plays crucial roles in general and site-specific

DSB repair, in telomere maintenance, and in mainte- missed due to lack of thorough examination of other

strains of NHEJ-deficient mice.nance of genomic stability.

A Potential Relationship between Ku80 Deficiency, Background mutations in various strains of NHEJ-deficient mice, either exacerbating or suppressing theDNA-PKcs Deficiency, and Aging

NHEJ has been proposed to play a causative role in the progeroid manifestations of NHEJ deficiencies, might

contribute to the apparently discrepant phenotypes ofaging process. DSBs are frequent events in mammalian

somatic cells, where they are also very commonly re- different lines. This possibility, which is relevant to any

gene deficiency/aging model, warrants further exami-paired by NHEJ. In addition, genetic studies support

the notion that NHEJ plays an important role in the re- nation and is discussed in other contexts below. Also,

given that both the Ku80- and DNA-PKcs-deficientpair of ROS-induced DNA lesions (Karanjawala and

Lieber, 2004). Since NHEJ can delete a few nucleotides mice show evidence of ongoing inflammation, poten-

tially indicative of infection, it is conceivable that someat sites of DSB repair, this process theoretically could

lead to accumulation of mutations and contribute to aging-like phenotypes might occur directly due to the

effects of chronic infection in the setting of immunode-cellular decline and aging (Karanjawala and Lieber,

2004). Moreover, in the absence of NHEJ, DSBs often ficiency, rather than due to impaired DNA repair. Os-

teopenia, for example, could result from elevated gluco-are repaired concomitant with large deletions and/or

translocations; thus, absent or even decreased levels corticoid levels induced by chronic physiologic stressfrom infection or malnutrition associated with intestinalof NHEJ also might contribute to accelerated aging.

The observation that phenotypes resembling ac- atrophy. The degenerative changes in afflicted strains

of DNA-PKcs- and Ku80-deficient mice affect only acelerated aging have been described in one strain each

of Ku80- and DNA-PKcs-deficient mice potentially sup- limited subset of organs. Therefore, either these NHEJ

factors are only involved in suppressing aging-relatedports this model. Thus, a line of Ku80-deficient mice

prematurely exhibits age-specific changes including changes in certain tissues or these degenerative changes

occur for reasons distinct from those that contribute toosteopenia, atrophic skin, liver lesions, and shortened

life span (Vogel et al., 1999). Likewise, a strain of DNA- aging in wild-type animals. Of note, degenerative changes

in these models occur in both highly proliferative (intes-PKcs-deficient mice recently has been noted to exhibit

age-related pathologies, with osteopenia, intestinal at- tine and skin) and relatively less proliferative (bone and

liver) tissues, apparently sparing many other tissues ofrophy, thymic lymphoma, and reduced longevity (Es-

pejel et al., 2004b). There are several potential explana- both types. These observations argue against a simple

relationship between mitotic status and dependence ontions to account for how NHEJ deficiency could cause

aging-like phenotypes. As described above, spontane- NHEJ in the suppression of aging.In addition to the above considerations, other modelsous DSBs in Ku80- or DNA-PKcs-deficient animals


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for NHEJ factor functions in suppressing aging may be 2004; Rothkamm et al., 2003; Takata et al., 1998). The

first step in HR is processing of DSBs by a nuclease topostulated. It is notable that efficiency of DSB repair

generate 3# ssDNA tails, which are coated with RPAmay decline with age in budding yeast (McMurray and

protein (Figure 4). The MRN complex, composed of theGottschling, 2003) and in mammalian tissues (Sedelni-proteins Mre11, Rad50, and Nbs1, is a candidate forkova et al., 2004; Singh et al., 2001), as well as in senes-this nuclease, although other nucleases are likely in-cent mammalian cells (Seluanov et al., 2004). Theoreti-volved as well. The MRN complex plays multiple othercally, such an age-related decline in DSB repair mightroles in DSB repair: it functions both upstream andcontribute to increased mutations and genomic re-downstream of ATM and has been proposed to pro-arrangements preferentially near the end of life, al-mote sister chromatid association and recombinationthough such a decline has not been rigorously proven.(Stracker et al., 2004). The Rad51 protein, assisted byAlso, loss of other functions of DNA-PKcs or Ku80,a number of factors including Rad52, Rad54, BRCA2,such as telomere end capping and/or DNA damage sig-and the Rad51 paralogs (XRCC2, XRCC3, Rad51B,naling, could contribute to aging phenotypes. In thisRad51C, and Rad51D), forms a nucleoprotein complexcontext, targeted inactivation of either Ku80 or DNA-with the DNA and directs the 3# ssDNA tails to searchPKcs, but not in Ligase IV or XRCC4, demonstratesout, invade, and pair with undamaged homologous se-synthetic lethality with ATM deficiency. These observa-quences. DNA polymerases then carry out repair usingtions argue for overlapping functions between Ku/DNA-the intact DNA as a template. The processes of DNAPKcs and ATM that do not involve classical NHEJ, per-strand exchange and extension generate Holliday junc-haps related to telomere maintenance (Sekiguchi et al.,tions (HJs), structures in which two dsDNA duplexes2001). Moreover, Ku80/Terc and DNA-PKcs/Terc doubleare intertwined. In mammalian DSB repair, HJs aredeficient mice show exacerbation of intestinal atrophythought to be resolved primarily via disengagement andand other aging-like phenotypes of Terc deficiency (Es-gap repair rather than cleavage of the HJ (Valerie andpejel et al., 2004a). Late generation Terc-deficient micePovirk, 2003).have an uncharacterized DSB repair defect (Wong etBRCA1 in HR and Other Cellular Processesal., 2000); loss of NHEJ in the context of dysfunctionalThe biology of the BRCA1 protein has proven to be verytelomeres would be predicted to further compromisecomplex; BRCA1 plays roles in multiple fundamentalDSB repair. In this regard, cells deficient in Ligase IVcellular processes. Biochemically, BRCA1, togetherand ATM or Terc and ATM show high levels of genomicwith its partner protein BARD1, possesses E3 ubiquitininstability and very rapid senescence (Sekiguchi et al.,ligase activity in addition to binding both DNA and2001; Wong et al., 2003), pointing to synergies betweenmultiple other proteins. Functionally, several lines ofDSB repair, DNA damage signaling, and telomere main-evidence link the BRCA1 protein to HR (Scully et al.,tenance in genomic maintenance and overall cellular vi-2004). BRCA1 is a phosphorylation target of ATM andability.the related PIKK, ATR, following DNA damage induc-In summary, there are many potential roles for NHEJtion. BRCA1 forms a complex with Rad51 and BRCA2in preventing premature aging-like phenotypes, such as(Dong et al., 2003) and can be detected with Rad51 inthose observed in Ku80- and DNA-PKcs-deficientS phase foci thought to represent stalled replicationmice. Current findings suggest, however, that agingforks. BRCA1 foci also form in response to ionizing ra-phenotypes likely do not result from a failure of DSBdiation and on chromosomes during meiotic recombi-

repair alone but instead from a loss of other functionsnation. BRCA1 deficiency leads to impaired HR-medi-

or combinations of functions of these proteins, perhapsated repair of chromosomal DSBs, hypersensitivity to

in concert with environmental factors, such as housingmany DNA-damaging agents, and genomic instability.

conditions and/or infection and/or background muta-The biochemical nature of BRCA1s involvement in HR

tions. Such complexities also may likely apply to otherremains unclear; one possibility is that BRCA1 may per-

DNA repair proteins implicated in the suppression ofform a scaffolding function, potentially coordinating the

aging as well, since, as we shall see, many of theseformation of functional repair complexes at DSBs. The

proteins play roles in other cellular processes besidesE3 ubiquitin ligase activity of BRCA1 may also be rele-

DNA repair and, as with NHEJ, only a subset of mutantsvant in this context; BRCA1 may modify other proteins

in any given DNA repair pathway show aging-related during HR to alter their functions or direct their degra-phenotypes.dation. Additionally, BRCA1 has been implicated in

many other functions outside HR: transcription, G2/MPotential Roles for Rad50 and BRCA1 checkpoint control, chromatin remodeling, and X inacti-in HR and Aging vation (Scully et al., 2004). It has also been proposedHR in DSB Repair that BRCA1 may play a role in NHEJ (Ting and Lee,Two factors with roles in HR, Rad50 and BRCA1 (breast 2004). Thus, BRCA1 plays roles in numerous cellularcancer susceptibility gene-1), appear linked to aging- processes, including DNA repair.like phenotypes in mouse models. HR is the other major BRCA1 and Rad50 Hypomorphspathway of DSB repair in mammalian cells. Unlike Show Aging PhenotypesNHEJ, HR uses the sister or (in some cases) the homol- Defects in Rad50 or BRCA1 cause progeroid pheno-ogous chromosome as a template to repair the broken types. Deficiency of Mre11, Nbs1, or Rad50 is not com-chromosome. patible with cellular survival (Stracker et al., 2004). How-

Whereas recent data indicate that NHEJ functions in ever, homozygosity for a Rad50 hypomorphic allele

DSB repair throughout the cell cycle, HR is largely re- (Rad50s/s

) permits viability; Rad50s/s

mice show a short-ened life span, cancer predisposition, and hemato-stricted to late S/G2 (Couedel et al., 2004; Mills et al.,


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Figure 4. HR at DSBs

See text for details. Reproduced by permission with modification from Oncogene (Valerie and Povirk, 2003), copyright 2003 Macmillan

Publishers Ltd.

poietic stem cell and spermatogenic failure (Bender et DNA repair roles may contribute to these aging-like mu-

tant phenotypes.al., 2002). Genomic instability is detectable in cells de-

rived from this animal. The attrition of the hema-

topoietic and male germ cell lineages occurs in large Single-Stranded DNA Lesions and Agingmeasure due to p53-mediated signaling triggered by Nucleotide Excision Repairgenomic instability (Bender et al., 2002). DNA lesions that affect only one DNA strand are re-

Homozyogous inactivation of BRCA1 results in early paired via BER or NER and its subpathways. Althoughembryonic lethality (Valerie and Povirk, 2003); however, BER is thought to play a critical role in the repair ofmice homozygous for a BRCA1 hypomorphic allele and oxidative lesions, mutations in genes involved in thishaploinsufficient for p53 (BRCA111/11/p53+/) are via- pathway do not produce aging manifestations: they areble and have many features reminiscent of accelerated either lethal or confer no obvious phenotypes (Hasty et

aging: wasting, skin atrophy, osteopenia, and malig- al., 2003). By contrast, lesions in some factors involvednancy (Cao et al., 2003b). There is compelling evidence in NER can lead to premature aging syndromes in micethat this phenotype results from p53-dependent re- and humans. NER is activated by a wide range of helix-sponses to unrepaired DNA damage (Cao et al., 2003b). distorting DNA lesions, including UV-induced photopro-Baseline p53 protein levels are higher in BRCA111/11/ ducts, bulky chemical adducts, and certain oxidativep53+/ mice than in p53+/ control animals. Similar to lesions. NER can be subdivided into two pathways,some NHEJ-deficient MEFs, BRCA111/11 MEFs show global genome NER (GG-NER) and transcription-cou-increased chromosomal abnormalities and premature pled NER (TC-NER), which differ with respect to thecellular senescence, and BRCA111/11 embryos show lesion detected and some of the factors involvedtissue SA--galactosidase activity, a marker of senes- (Mitchell et al., 2003; Figure 5).cence. Thus, in both the Rad50s/s and BRCA111/11/ The basic NER machinery consists of the proteins

p53+/ mice, signs of premature aging occur as a con- XPA through XPG, the CSA and CSB proteins, and

sequence of the p53-mediated responses to unrepaired other participants such as the basal transcription factor

DNA damage. However, since BRCA1 and Rad50 are TFIIH. The GG-NER specific factors, XPC (in a complex

both involved in multiple cellular processes, the possi- with the HR23B protein) and XPE are responsible fordetecting helix-distorting lesions that occur throughoutbility exists that loss of other functions beyond their


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Figure 5. Global-Genome NER and Tran-

scription-Coupled NER

Factors with aging-related mutant pheno-

types (XPD, CSA, CSB, ERCC1, and XPF) are

shown in red. See text and Table 1 for details.

Reprinted with modification from Mitchell et al.

(2003) with permission from Elsevier.

the genome. The TC-NER specific factors, CSA and fective in these individuals. Mice bearing a targeted

mutation in XPD that recapitulates a human TTD muta-CSB, are involved in repair specifically in transcribed

regions and, when RNA polymerase II stalls at a lesion, tion show similar manifestations to human TTD patients.

Additionally, these XPDTTD mice show aging-associatedcontribute to displacing the stalled polymerase to cre-

ate access for repair machinery. Following recogni- changes such as wasting, scoliosis, osteoporosis, and

melanocyte loss (de Boer et al., 2002). XPDTTD/XPAtion of the damaged DNA, common NER factors are

recruited in both GG-NER and TC-NER. TFIIH, which double mutants show a much more rapid degenerative

phenotype, suggesting that in the setting of impairedcontains two DNA helicases, XPB and XPD, unwinds

the DNA flanking the lesion. The single-strand DNA transcription and/or TC-NER, a total lack of NER is ex-

tremely deleterious. Similarly, mice deficient in both(ssDNA) binding protein RPA binds to and stabilizes the

unwound DNA strands, and XPA aids in lesion recogni- CSB and XPA also die within a few weeks after birth,

although only cerebellar defects have been describedtion. Two structure-specific endonucleases, XPG and

XPF (the latter in a complex with the protein ERCC1), in detail in these animals (Murai et al., 2001).The aging-like phenotypes in human CS patients andthen make single-strand incisions on either side of the

lesion to release an oligonucleotide. The resulting gap in XPDTTD, XPDTTD/XPA, and CSB/XPA mouse mutants

may be explained by the fact that a failure to repairis filled by template-dependent DNA polymerization fol-

lowed by ligation. lesions in transcribed genes can result in cell death,

leading to tissue attrition and aging. Stalled RNA poly-Several core NER factors have been implicated in

processes aside from NER. XPB and XPD are critical in merase provides a signal for activation of p53-depen-

dent apoptosis (Ljungman and Lane, 2004). In this re-RNA polymerase II transcription; CSB associates with

RNA polymerases I and II; and XPF/ERCC1 has been gard, it will be of interest to determine whether p53

deficiency rescues the aging-like features of XPDTTD,implicated in repair of interstrand crosslinks, homology

directed repair, and processing of the 3# G strand over- XPDTTD/XPA, and CSB/XPA mice. Alternatively, the in-

volvement of XPD and the CSB proteins in transcriptionhang at telomeres.

Some NER Defects Lead to Premature suggests that impaired transcription of critical genes

may play a role in causing these progeroid phenotypes,Aging Phenotypes

Numerous human patients and mouse strains with de- perhaps interacting with the repair defects in a compli-cated fashion.fects in different NER factors exist, and some have phe-

notypes reminiscent of premature aging (Mitchell et al.,

2003). Defects in the TC-NER-specific factors CSA or WRN and Aging

Defects in proteins with less well-defined functions inCSB lead to Cockayne syndrome in humans, a severely

debilitating disorder with striking progeroid features. DNA repair can lead to aging phenotypes as well. The

human disease WS represents the best model for pre-CSA- and CSB-deficient mice show much milder phe-

notypes than their human counterparts (Mitchell et al., mature aging in humans (Goto, 1997). WS patients

develop premature graying, cataract, loss of subcuta-2003). Patients with specific mutations in XPD suffer

from trichothiodystrophy (TTD), a disease character- neous fat, skin atrophy, osteoporosis, diabetes, athero-

sclerosis, and malignancies. WS cells senesce prema-ized by photosensitivity, brittle hair, skin defects, and

a shortened life span. These patients show defects in turely in culture. The gene defective in WS, WRN,

encodes a helicase of the RecQ family (a group definedtranscription of hair- and skin-specific transcripts (and

perhaps other mRNAs) (Bergmann and Egly, 2001). In by its similarity to E. coli RecQ helicase). WRN also

possesses an exonuclease domain. WRN plays a roleaddition, cells derived from TTD patients show NER de-fects, suggesting that multiple functions of XPD are de- in the maintenance of overall genomic stability, and


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Review507

WRN may be involved in multiple DNA repair pathways is an important cause of mortality in yeast when life

span is measured in this fashion (Sinclair and Guarente,(Bachrati and Hickson, 2003).

1997). Loss of Sir2 increases rDNA recombination andRecent experiments using mice bearing targeted mu-

shortens life span; whereas an extra genomic copy oftations in WRN provide evidence that, with respect toSir2, which increases rDNA stability, extends life spanaging, the most relevant sites of WRN function are the(Kaeberlein et al., 1999).telomeres. WRN-deficient mice do not recapitulate hu-

It has been proposed that Sir2 activity ties the energyman WS (Bachrati and Hickson, 2003). Based on thestatus of the yeast cell to longevity (Blander and Guar-observations that human WS cells show telomeric in-ente, 2004; Lin et al., 2000). When nutrients are scarce,stability (Schulz et al., 1996; Tahara et al., 1997) andyeast cells preferentially employ respiration rather thanthat introduction of telomerase into WS fibroblasts canfermentation to generate ATP (Lin et al., 2002). Thisrescue their premature senescence (Wyllie et al., 2000),metabolic switch alters the metabolism of the cell,it was proposed that WRN-deficient mice may not de-increasing the NAD/NADH ratio and/or decreasingmonstrate a strong phenotype due to the abundantlevels of the Sir2 inhibitor nicotinamide, in turn activa-mouse telomere reserve (Lombard et al., 2000). This hy-ting Sir2 and increasing rDNA stability (reviewed inpothesis has been proven correct by the generation ofBlander and Guarente, [2004]). Strikingly, overexpres-mice deficient in both WRN and Terc (Chang et al.,sion or pharmacologic activation of Sir2 in worms and2004; Du et al., 2004). In the WRN/Terc double deficientflies also extends life span (Rogina and Helfand, 2004;animals, phenotypes reminiscent of human WS that areTissenbaum and Guarente, 2001; Wood et al., 2004).not observed in either single mutant are present: os-Sir2-driven increased longevity in C. elegans requiresteopenia, diabetes, and sarcomas. Phenotypes ordinar-the Daf-16 transcription factor (Tissenbaum and Guar-ily seen in late-generation Terc-deficient animals alsoente, 2001). Daf-16 is a critical mediator in the insulin-occur earlier in the compound mutants and are associ-like signaling pathway, normally employed by worms toated with a greater degree of telomeric dysfunction.arrest as extremely long-lived larvae under unfavorableWhether or not these phenotypes depend on p53 func-environmental conditions; certain mutations in thistion is unknown; the premature senescence of humanpathway confer longevity upon adult worms. The mech-WS fibroblasts is p53 dependent (Davis et al., 2003).anisms by which Sir2 extends life span in flies are cur-The latter observation suggests that cellular checkpointrently unclear. Sir2 family members may play a generalfunctions may be involved in producing the clinical fea-role in mediating caloric restriction (Sohal and Wein-tures of WS.druch, 1996), an intervention capable of extending lifeThe exact role of WRN in telomere maintenance isspan in many different organisms from yeast to mam-currently unclear. WRN can be detected at the telo-mals (Cohen et al., 2004; Howitz et al., 2003; Lin et al.,meres in the absence of telomerase function in mam-2000; Rogina and Helfand, 2004; Wood et al., 2004),malian cells (Johnson et al., 2001; Opresko et al., 2004),although in yeast the involvement of Sir2 in CR appearsand Sgs1p, the S. cerevisiae WRN homolog, is requiredto be strain specific (Kaeberlein et al., 2004).for recombinational telomere maintenance in telom-

In mammals, there are seven Sir2 family members,erase-deficient cells in yeast (Cohen and Sinclair, 2001;designated SIRT1SIRT7 (Frye, 2000); SIRT1 is theHuang et al., 2001; Johnson et al., 2001). WRN also in-most highly related to S. cerevisiae Sir2. The role ofteracts with the telomeric protein TRF2 and can unwindSIRT1 in mammalian longevity has not yet been directly

and degrade telomeric D loop structures. These obser-tested, since on a pure strain background SIRT1-defi-

vations suggest that WRN may play a role in providingcient animals die very early as a consequence of

telomeric access to other factors involved in telomeremultiple developmental defects (Cheng et al., 2003;

maintenance (Machwe et al., 2004; Opresko et al., 2004;McBurney et al., 2003). Unlike yeast Sir2, which has no

Orren et al., 2002). Overall, the phenotype of the WRN/known targets aside from histones, SIRT1 possesses a

Terc double knockout mouse argues that defectivelarge and growing list of targets, some of which, includ-

telomere maintenance is an important factor in produc-ing p53 and forkhead transcription factors (mammalian

ing the premature aging-like aspects of WS in humans,homologs of Daf-16), modulate cellular resistance to

although it does not exclude a role for other functionsoxidative and genotoxic stress (Blander and Guarente,

of WRN at nontelomeric sites. 2004). Additionally, SIRT1, like Sir2, has recently beenshown to directly modify chromatin and silence tran-

Sir2: A Link between Metabolism, Genome scription (Vaquero et al., 2004). It is now important toStability, and Life Span determine whether SIRT1, in addition to silencing tran-In the models discussed above, decreased genomic scription, also suppresses recombination and genomicstability is associated with shortened life span. The Sir2 instability via chromatin effects and if so, whether suchfamily of proteins provides an example in which in- an activity could be involved in regulating aging increased genomic stability extends life span (Blander mammals. SIRT1 conditional alleles may allow studiesand Guarente, 2004). In S. cerevisiae, the chromatin of the role of this protein in aging.regulatory factor Sir2 (silent information regulator-2) The true mammalian functional ortholog of Sir2, iffunctions as an NAD-dependent histone deacetylase one exists, might also be a different mammalian Sir2(Imai et al., 2000) to suppress recombination and turns family member (or members) than SIRT1. SIRT2 andoff transcription at multiple genomic loci (Blander and SIRT3 are unlikely to play this role, because these pro-Guarente, 2004). One metric of aging in yeast is the teins are cytoplasmic and mitochondrial, respectively,

number of divisions that a single mother cell un- rather than chromatin associated (Blander and Guar-ente, 2004). Thus far, no information has been forth-dergoes. The excision and replication of rDNA circles


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Figure 6. A Model for the Role of Unresolved

DNA Lesions in Aging

Unrepaired DNA lesions can activate the cell

cycle checkpoint machinery, leading to se-

nescence or apoptosis and subsequent cel-

lular attrition and tissue dysfunction. Some

types of unrepaired DNA damagelike DNADSBscan trigger genomic instability, which

can in turn lead to further DNA damage. In

addition, unrepaired DNA damage can di-

rectly compromise cellular processes like

transcription, an effect that could also impair

tissue function. Such unrepaired DNA dam-

age will be frequent in DNA repair mutants

and lead to accelerated degenerative changes;

in wild-type organisms unrepaired damage is

infrequent, owing to efficient repair systems.

coming regarding the functions of the four remaining cussed above, environmental factors including housing

conditions, infectious agents, diet, and many other in-SIRTs, SIRT4SIRT7, but these remain candidates for

fluences, also likely play a significant role in the expres-proteins that may regulate longevity through genome

sion of aging phenotypes in mouse models and, per-stabilization.haps, in humans as well. Thus, aging is likely the

outcome of a complex interplay between the geneticConclusions

endowment of an organism and the stresses placedThe hypothesis that nuclear DNA, a critically important

upon it by its particular environment.cellular constituent that cannot be replaced, is an im-

All mouse models that link DNA repair to aging pos-portant target of age-related change is supported by

sess defects in DNA repair and have shortened lifeevidence that nuclear DNA damage and mutations ac-

spans. It is important to bear in mind the potential pit-cumulate with age. While ROS are likely to be one im-

falls of such models (Hasty and Vijg, 2004; Miller, 2004).portant source of this damage, there are numerous

Aging encompasses a wide spectrum of degenerative

other cellular and environmental sources of damage, processes, many of which are quite nonspecific, bothand the impact of such lesions may be enhanced byclinically and pathologically (Harrison, 1994). Thus, it is

age-related compromise of DNA repair. In the latterdifficult to arrive at a strict, experimentally useful defini-

context, most premature aging syndromes are causedtion of aging. Factors implicated in organismal decline

by mutations in genes encoding proteins involved inin genetic models might not play a role in the normal

DNA repair (Karanjawala and Lieber, 2004). Accumula-aging processes. A related difficulty is that premature

tion of mutations in critical genes may be one generalaging models fail to recapitulate all aspects of aging

mechanism by which compromised DNA repair couldbut are instead segmental progerias (Hasty and Vijg,

contribute to aging. In addition, p53-mediated senes-2004; Miller, 2004); that is, they reproduce in an ac-

cence and apoptosis, in response to DNA damage, alsocelerated fashion some but not all aspects of aging as

likely contribute to aging (Figure 6). Indeed, the fact that it occurs in wild-type animals. In this rega rd, the myriadlesions in several disparate repair systems cause phe- histopathologic changes of normal aging (Table 2) cor-notypes that are broadly similar to one another (Table 1) respond poorly with the changes that occur in modelsis consistent with the notion that the specific chemical of premature aging (Table 1). Mammalian aging is not

nature of the accumulated DNA lesions may be less likely a single process but rather the decline of manyimportant than their ability to activate the common cel- somatic functions, heavily influenced by the environ-lular checkpoint machinery. ment; this is a complex interplay that is extremely diffi-

It remains unclear why only certain DNA repair mu- cult to model accurately. For these reasons, genetictants in particular pathways show progeroid pheno- models of extended life span are likely to be more infor-types. In some cases, it may simply be that some mu- mative than models with reduced longevity with re-tants have not been scrutinized sufficiently to reveal spect to physiologically relevant causes of decline andsuch effects. However, genetic background effects al- mortality; in such long-lived organisms, life span-limit-most certainly play an important role in modifying the ing factors must of necessity be counteracted.aging-like manifestations of DNA repair deficiencies. There are many outstanding questions regarding theAlso, it must be remembered that many of the DNA re- connection between DNA repair and aging that willpair genes and factors implicated in suppressing aging benefit from the application of emerging techniques inalso play roles in cellular processes other than DNA re- molecular biology and genetics. In models of prematurepair, and therefore aging-like phenotypes might be en- aging, the most vulnerable system fails first, leading to

hanced by impairment of other cellular functions in death and precluding gain of insights from effects onother, potentially more relevant, organ systems. Thisconjunction with altered DNA repair. In addition, as dis-


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Review509

chondrial DNA is inversely related to maximum life span in the heartdifficulty might be overcome with hypomorphic allelesand brain of mammals. FASEB J. 14, 312318.or via conditional knockouts of relevant genes. The lat-Barzilai, A., Rotman, G., and Shiloh, Y. (2002). ATM deficiency andter approach, for example, might allow an evaluation ofoxidative stress: a new dimension of defective response to DNAthe roles of genes essential during embryogenesis indamage. DNA Repair (Amst.) 1, 325.

the aging process. Such an approach also could permitBassing, C.H., and Alt, F.W. (2004). The cellular response to general

insights into the types of DNA lesions that are mostand programmed DNA double strand breaks. DNA Repair (Amst.)important in causing aging in different tissues by allow- 3, 781796.

ing impairment of specific DNA repair pathways in de-Ben-Porath, I., and Weinberg, R.A. (2004). When cells get stressed:

fined cell populations. For example, tissue-specific de- an integrative view of cellular senescence. J. Clin. Invest. 113, 813.letion of DNA damage-response/checkpoint genes in Bender, C.F., Sikes, M.L., Sullivan, R., Huye, L.E., Le Beau, M.M.,DNA repair mutants showing evidence of premature Roth, D.B., Mirzoeva, O.K., Oltz, E.M., and Petrini, J.H. (2002). Can-

cer predisposition and hematopoietic failure in Rad50(S/S) mice.aging might allow the direct effects of these DNAGenes Dev. 16, 22372251.lesions to be distinguished from the cellular responsesBergmann, E., and Egly, J.M. (2001). Trichothiodystrophy, a tran-to them. Further insights into the basic biology of DNAscription syndrome. Trends Genet. 17, 279286.repair proteins, particularly regarding the exact natureBlander, G., and Guarente, L. (2004). The Sir2 family of protein de-of the DNA lesions that these repair systems respondacetylases. Annu. Rev. Biochem. 73, 417435.to and their roles in checkpoints, may also shed moreBronson, R.T., and Lipman, R.D. (1991). Reduction in rate of occur-light on the role of various DNA lesions in contributingrence of age related lesions in dietary restricted laboratory mice.

to aging.Growth Dev. Aging 55, 169184.

Eventually, the importance of DNA damage in agingBrowne, S.E., Roberts, L.J., 2nd, Dennery, P.A., Doctrow, S.R., Beal,might be directly tested via the generation of experi-M.F., Barlow, C., and Levine, R.L. (2004). Treatment with a catalytic

mental organisms with enhanced efficiency of DNA antioxidant corrects the neurobehavioral defect in ataxia-telangiec-maintenance, which would be predicted to show re- tasia mice. Free Radic. Biol. Med. 36, 938942.

tarded aging. Although the plethora of different repair Campisi, J. (2002). Between Scylla and Charybdis: p53 links tumorsuppression and aging. Mech. Ageing Dev. 123, 567573.systems makes this a challenging, if not impossible,

undertaking, the existence of Sir2, a regulator of ge- Cao, J., Venton, L., Sakata, T., and Halloran, B.P. (2003a). Expres-sion of RANKL and OPG correlates with age-related bone loss innome stability and aging in yeast, offers encourage-male C57BL/6 mice. J. Bone Miner. Res. 18, 270277.ment for those seeking similar global regulators ofCao, L., Li, W., Kim, S., Brodie, S.G., and Deng, C.X. (2003b). Senes-genome maintenance and potentially DNA repair incence, aging, and malignant transformation mediated by p53 inmammals.mice lacking the Brca1 full-length isoform. Genes Dev. 17, 201213.

Chang, S., Multani, A.S., Cabrera, N.G., Naylor, M.L., Laud, P., Lom-

bard, D., Pathak, S., Guarente, L., and DePinho, R.A. (2004). Essen-Acknowledgments

tial role of limiting telomeres in the pathogenesis of Werner syn-

drome. Nat. Genet. 36, 877882.The authors would like to thank members of the Alt lab, as well asCheng, H.L., Mostoslavsky, R., Saito, S., Manis, J.P., Gu, Y., Patel,Roderick Bronson, Ronny Drapkin, Toren Finkel, Lenny Guarente,P., Bronson, R., Appella, E., Alt, F.W., and Chua, K.F. (2003). Devel-Marcia Haigis, F. Bradley Johnson, Michael Lieber, Kevin Mills,opmental defects and p53 hyperacetylation in Sir2 homologRalph Scully, Peter Sorger, Tony Wynshaw-Boris, and especially(SIRT1)-deficient mice. Proc. Natl. Acad. Sci. USA 100, 10794Ned Sharpless for helpful discussions and comments on the manu-10799.script. F.W.A. is an Investigator of the Howard Hughes Medical In-

stitute. This work was supported by an Ellison Foundation Senior Cohen, H., and Sinclair, D.A. (2001). Recombination-mediated

Scholar Award (to F.W.A), a K08 award from NIA/NIH (to D.B.L.), lengthening of terminal telomeric repeats requires the Sgs1 DNA

a Pfizer Postdoctoral Fellowship in Immunology/Rheumatology (to helicase. Proc. Natl. Acad. Sci. USA 98, 31743179.

K.F.C.), a Senior Postdoctoral Fellowship from The Leukemia and Cohen, H.Y., Miller, C., Bitterman, K.J., Wall, N.R., Hekking, B., Kess-Lymphoma Society (to R.M.), and a Long-Term Fellowship from the ler, B., Howitz, K.T., Gorospe, M., de Cabo, R., and Sinclair, D.A.European Molecular Biology Organization (to S.F.). (2004). Calorie restriction promotes mammalian cell survival by in-

The authors would like to apologize to those whose work was ducing the SIRT1 deacetylase. Science 305, 390392.not cited, due to space constraints. We have referenced other arti-

Couedel, C., Mills, K.D., Barchi, M., Shen, L., Olshen, A., Johnson,cles in this issue and recent in-depth reviews that provide these

R.D., Nussenzweig, A., Essers, J., Kanaar, R., Li, G.C., et al. (2004).references.

Collaboration of homologous recombination and nonhomologous

end-joining factors for the survival and integrity of mice and cells.

Genes Dev. 18, 12931304.

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